This project provides the first step toward the long term goal of developing an enzyme drug for commercialization acting on the extracellular matrix to enhance the efficacy of broad range of cancer therapeutics in solid tumors. The extracellular matrix (ECM) surrounding all cells is crucial for biological functions in tissues throughout the body. In solid tumors, the extracellular matrix can limit the ability of cancer drugs to penetrate and act on cancer cells. In this project we propose to engineer an enzyme that acts to specifically degrade the ECM in tumors, allowing cancer drugs to penetrate and act in tumors more effectively. Degradation of the ECM of solid tumors has been demonstrated both preclinically and clinically to to enhance a range of cancer drugs, including potentially traditional chemotherapeutics and newer agents such as antibodies and immuno-oncology agents. Previously used ECM degrading enzymes are not specific for tumors and degrade the ECM in peripheral tissues as well, leading to well-characterized, mechanistic, adverse events. These adverse events limit the dose of the ECM degrading enzymes which compromises the efficacy of the co-administered cancer drugs. This project proposes to target the activity of the ECM degrading enzyme to the tumor microenvironment, increasing the efficacy of co-administered cancer drugs, while also decreasing peripheral ECM degradation and reducing adverse events. To design this enzyme we will first use a computational approach to analyze the enzyme's structure and predict how changes in the enzyme might target enzyme action to the distinct conditions found associated with cancer in the tumor microenvironment. Second, based on these predictions we will create different versions of the target enzyme and test their activity in conditions representing the environment in tumors compared with conditions found in normal tissue, verifying that the predicted changes in the enzyme produce the desired activity specifically targeting tumors. Third, when a version, or versions, of the enzyme are found to specifically target the tumor microenvironment, we will produce larger quantities of this enzyme in preparation for future studies that will characterize the action of the enzyme in both cancer models, testing enhanced efficacy, and in safety models demonstrate the decrease of mechanistic adverse effects. The proposed outcome of this project will be an enzyme (or enzymes) suitable for preclinical pharmacology studies, the first step in producing a clinical candidate that would ultimately advance into testing in clinical trials, targeting pancreatic cancer and other solid tumors in which the extracellular matrix limits the action of cancer drugs.